1. Field of the Invention
This invention relates generally to the fields of Raman spectroscopy and spectroscopic imaging. Specifically, the present invention relates to dual- and multilayered substrates useful in surface enhanced Raman spectroscopic applications.
2. Description of the Related Art
Surface enhanced Raman scattering (SERS) has been used to monitor the presence of chemical species for a wide variety of applications, including: medicine, biochemical research), electrochemistry ultrahigh vacuum science (UHV) environmental monitoring and many others (1–11). The main reason for its widespread application arises from its ability to provide structural information about the analyte being measured, while also permitting trace analyses, i.e., single molecule/particle detection in some cases (12–14). Detection of trace amounts of analyte is due to the large signal enhancements achieved with SERS, typically 103–106, as compared to spontaneous Raman scattering. While an exact description of the SERS phenomenon is complex and not completely understood, it is well known that the large SERS enhancement factors, achieved by placing the analyte of interest in contact with a roughened metal surface, occur primarily through a combination of a chemical enhancement mechanism and an electromagnetic enhancement mechanism (15–17).
Chemical enhancement arises from the interaction of the analyte with the metal surface upon adsorption. This results in a charge transfer between the metal and adsorbate, resulting in an overlap of the transition wave functions of the metal and analyte. Therefore, for chemical enhancement, direct contact between the metal surface and the analyte is required. Electromagnetic enhancement is attributed to an increased electromagnetic field near the metal surface due to surface plasmon resonance (SPR). Upon excitation of the substrate with the appropriate wavelength of light, metal conduction electrons are excited to collective oscillation by the incident oscillating electromagnetic wave of excitation light. This oscillation provides an increased interaction between the metal surface and the electron cloud of the analyte molecule and is believed to account for the majority of the signal enhancement for most analyses (18).
In an attempt to take advantage of the potentially large signal enhancement factors associated with SERS, many different types of substrates have been developed. These substrates are typically made of silver, gold or copper and, in rare cases, alkali and transition metals (19–20). Some of the most commonly employed SERS substrates include noble metal colloids (21–22) electrochemically roughened electrodes (23–25), acid-etched metal foils (26), chemically produced silver island films (27–29) vapor deposited metal island films (30), and silver films over nanoparticles/nanostructures (SFONnn) (9, 31–34).
Metal colloids typically provide the greatest SERS enhancements factors. In fact, in specific cases, colloidal silver substrates have been reported to be capable of single molecule detection (12–13, 35). However, SERS analyses performed using these metal colloidal substrates demonstrate poor stability over time, as the colloidal particles tend to aggregate and precipitate in solution, thereby changing the morphological as well as chemical properties of the substrate. In addition, inhomogeneous enhancement is present throughout the sample due to the random distribution and aggregation of the colloidal particles.
Metal island film substrates are capable of providing significant SERS enhancement factors, while reducing the problems associated with aggregation and precipitation in metal colloidal substrates. Metal island film substrates provide a significantly greater spot-to-spot reproducibility than colloidal substrates and are relatively simple to fabricate. Unfortunately, enhancements obtained from this type of substrate are significantly less than those obtained from colloidal substrates and analytical studies are complicated by the need to accurately control the deposition parameters that influence the islands' size, shape and spacing distribution (36). Recently, a method of preparing dual-layered metal island film substrates that are capable of significant signal enhancements relative to conventional single layer silver island films, making them much more sensitive has been developed and characterized (37). Substrates were prepared by coating 75 nm of silver on a surface to form a layer of discrete silver islands. The silver islands were exposed to air and a second silver island layer of a 45 nm thickness was deposited on top of the previous layer. Scanning electron microscope images of these substrates revealed that the second layer of silver islands were deposited selectively on underlying silver islands creating an optimal surface morphology, both size and shape, for SERS. Using these substrates, SERS signal enhancement factors of 104, as compared to spontaneous Raman scattering, were achieved.
A three-layered substrate that combines aspects of colloidal substrates with the lifetime stability and reproducibility of silver film over nanostructure (SFON) based substrates has been developed (38–39). The substrates were prepared by placing 12 nm diameter colloidal gold (Au) nanoparticles on a glass support and chemically coating them with silver (Ag) before thermally evaporating a layer of silver islands on top of the chemically deposited silver. This process created substrates that had well controlled and distributed colloidal particles with a silver coating from the chemical deposition as well as silver islands between the colloidal particles to reduce fluorescence background signals from impurities deposited during the chemical reduction process. These substrates also exhibited enhancement factors on the order of 7×104, as well as an enhanced reproducibility of 15%, compared to colloidal substrates. In both of these previous studies, multiple layers of noble metals were used to carefully construct roughened metal surfaces with precisely controlled surface morphologies which were attributed as the primary reason for the enhanced electromagnetic field and the increased reproducibility.
Accordingly, a need in the art is recognized for improved surface enhanced Raman spectroscopic substrates effective to increase a SERS signal. More specifically, the prior art is deficient in surface enhanced Raman spectroscopic substrates comprising at least two or more continuous SERS-active metal layers deposited thereon. The present invention fulfills this longstanding need and desire in the art.